Triplex targeting of a native gene in permeabilized intact cells: covalent modification of the gene for the chemokine receptor CCR5.
A 12 nucleotide oligodeoxyribopurine tract in the gene for the chemokine receptor CCR5 has been targeted and covalently modified in intact cells by a 12mer triplex forming oligonucleotide (TFO) bearing a reactive group. A nitrogen mustard placed on the 5'-end of the purine motif TFO modified a guanine on the DNA target with high efficiency and selectivity. A new use of a guanine analog in these TFOs significantly enhanced triplex formation and efficiency of modification, as did the use of the triplex-stabilizing intercalator coralyne. This site-directed modification of a native chromosomal gene in intact human cells under conditions where many limitations of triplex formation have been partially addressed underscores the potential of this approach for gene control via site-directed mutagenesis.
Belousov
E S
ES
Epoch Pharmaceuticals Inc., 1725 220th Street S.E., #104 Bothell, WA 98021, USA.
Afonina
I A
IA
Kutyavin
I V
IV
Gall
A A
AA
Reed
M W
MW
Gamper
H B
HB
Wydro
R M
RM
Meyer
R B
RB
D000477:Alkylating Agents; D001483:Base Sequence; D001600:Berberine Alkaloids; D002460:Cell Line; D004247:DNA / Q000737:chemistry* / Q000235:genetics*; D018390:Gene Targeting; D006147:Guanine / Q000737:chemistry; D006801:Humans; D007364:Intercalating Agents; D008466:Mechlorethamine / Q000737:chemistry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics; D016133:Polymerase Chain Reaction; D019713:Receptors, CCR5 / Q000235:genetics*; D014162:Transfection
D000477:Alkylating Agents; D001600:Berberine Alkaloids; D007364:Intercalating Agents; D009838:Oligodeoxyribonucleotides; D019713:Receptors, CCR5; D008466:Mechlorethamine; D006147:Guanine; C000666:coralyne; D004247:DNA